Monoclonal antibodies have a great therapeutic potential and play an important role in today's medical portfolio. During the last decade, a significant trend in the pharmaceutical industry has been the development of monoclonal antibodies (mAbs) and antibody Fc-fusion polypeptides (crystallizable fragment-fusion polypeptides) as therapeutic agents across diverse clinical settings including oncology, chronic inflammatory diseases, transplantation, infectious diseases, cardiovascular medicine, or ophthalmologic diseases (Carter, J. P., Nature Reviews Immunology 6 (2006) 343-357; Chan, A. C. and Carter, J. P., Nature Reviews Immunology 10 (2010) 301-316).
The clinical efficiency of a therapeutic antibody relies mainly on two functionalities: i) the target-specific binding mediated by the Fv-domain, and ii) the immune-mediated effector function such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity), and ADCP (antibody-dependent cellular phagocytosis) which are mediated by the antibody Fc-region. The Fc-region of an immunoglobulin of the IgG class comprises the hinge region and two constant domains (CH2 and CH3). The Fc-region also interacts with the neonatal FcRn receptor and thereby determines the half-life of the antibody in vivo. The hinge region is the region at which the arms of an antibody molecule form a Y-like structure enabling flexibility in the molecule at this point. The IgG subclass/subclasses differ in the number of disulfide bonds and the length of the hinge region.
The effector functions associated with the Fc-region of an antibody vary with the class and subclass of the antibody and include e.g. binding of the antibody via its Fc-region to a specific Fc receptor (FcR) on a cell which triggers various biological responses (see e.g. Jiang, X.-R., et al., Nature Reviews Drug Discovery 10 (2011) 101-110; Presta, L. G., Current Opinion in Immunology 20 (2008) 460-470).
The hinge region of an antibody or of an Fc-region comprising fusion polypeptide or conjugate is involved in at least a part of the antibody's functions such as antigen binding and Fc-region-mediated antibody effector functions. Whereas antigen binding (especially bivalent avid antibody binding) depends on the flexibility, length and spatial orientation of a particular/native hinge region the Fc-region mediated effector functions are dependent on the class and subclass of the antibody. The functional monovalency observed for some human IgG4 antibodies in comparison with the bivalency for the other IgG antibodies is another example showing the involvement of the Fc-region region in antigen binding properties (Salfeld, J. G., Nature Biotechnology 12 (2007) 1369-1372; Presta, L. G., Current Opinion in Immunology 20 (2008) 460-470).
Levary, D. A., et al., report protein-protein fusion catalyzed by Sortase A (PLOS ONE 6 (2011)). Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase A-mediated protein ligation is reported by Madej, M. P., et al. (Biotechnol. Bioeng. 109 (2012) 1461-1470). Ta, H. T., et al., report enzymatic single-chain antibody tagging as a universal approach to targeted molecular imaging and cell homing in cardiovascular diseases (Cir. Res. 109 (2011) 365-373). Popp, M., et al., report making and breaking peptide bonds—protein engineering using sortase (Angew. Chem. Int. Ed. Eng. 50 (2011) 5024-5032). In WO 2010/087994 methods for ligation and uses thereof are reported.